Formation of the cardiac outflow tract (OFT) is a particularly important aspect of cardiogenesis: the dimensions, orientation, and subdivision of the OFT are crucial for effective transport of blood from the heart to the periphery. OFT development initiates with the assembly of a small tube of myocardium that is appended to the arterial pole of the embryonic ventricle. Construction of this tube requires the selection of late- differentiating progenitor cells from the second heart field (SHF), recruitment of the selected cells to the arterial pole, and the organization of these cells into the appropriate three-dimensional configuration. Although several signals have been implicated in regulating SHF differentiation, we still do not understand the full roster of factors responsible for producing th correct number of OFT cardiomyocytes within an appropriate timeframe, nor do we understand how the OFT cells align themselves with the functioning heart to create a tract of the proper dimensions. In this project, we exploit the utility of the zebrafish as a model organism in order t elucidate the mechanisms regulating OFT development. Notably, our recent work in zebrafish has shed light on the mechanisms controlling the number of OFT cells by revealing an intriguing role for the cell adhesion molecule Cadm4 in limiting the production of OFT progenitors. Additionally, our preliminary studies have provided new insight into the regulation of OFT morphogenesis by suggesting an important influence of physical forces on the dimensions of the OFT. Here, we propose to extend both of these lines of investigation in order to establish novel paradigms for the control of OFT size and shape. In Aim 1, we will employ loss-of-function and gain-of- function strategies, evaluation of chimeric embryos, and structure-function analysis in order to test a model in which extracellular interactions between Cadm4 and Cadm3 act within the SHF to regulate the proliferation of progenitor cells and thereby limit their contributin to the OFT. In addition, considering the impact of Cadm family members on proliferation and differentiation, we will test whether these molecules influence the same cell behaviors during the process of myocardial regeneration. In Aim 2, we will determine the impact of cardiac function on OFT architecture, using morphometric analysis, live imaging, and modulation of multiple distinct aspects of cardiac function. These strategies will allow us to test a model in which blood flow induces expansion of the endocardial lumen within the OFT, creating a scaffold that sets the dimensions for the OFT myocardium. Together, these experiments will illuminate the network of pathways that collaborate to insure the appropriate morphology of the embryonic OFT. In the long term, an improved understanding of the mechanisms that control the initial dimensions of the OFT is likely to have a substantial impact on the comprehension and treatment of congenital heart disease.